Lactoferrin (Lf) is an iron-binding multi-functional glycoprotein which has numerous physiological functions such as iron transportation, anti-microbial activity and immune response. In this study, different in silico approaches were exploited to investigate Lf protein properties in a number of mammalian species. Results showed that the iron-binding site, DNA and RNA-binding sites, signal peptides and transferrin motifs in the Lf structure were highly conserved. Examined sequences showed three conserved motifs which were repeated twice in the Lf structure, demonstrating ancient duplication events in its gene. Also, results suggest that the functional domains in mammalian Lf proteins are Zinc finger, Tubulin/FtsZ, GTPase, a/b hydrolase and Zinc knuckle. The potential site for nucleic acid binding and the major DNA and RNAbinding sites in this protein were found in the lactoferricin (Lfc) fragment. Due to its high positive charge, Lf is able to bind a large number of compounds. Our analysis also revealed that the interactions between Lf and ITLN1, LYZ, CSN2, and CD14 proteins played an important role in the protective activities of Lf. Analysis for the prediction of secondary structures indicated that high amounts of a-helix, b-strand and b-sheet were present in Lf. The high degree of conservation among mammalian Lf proteins indicates that there is a close relationship between these proteins, reflecting their important role.